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Abstract

Background

Viral respiratory infections are common worldwide and range from completely benign
disease to life-threatening illness. Symptoms can be unspecific, and an etiologic
diagnosis is rarely established because of a lack of suitable diagnostic tools. Improper
use of antibiotics is common in this setting, which is detrimental in light of the
development of bacterial resistance. It has been suggested that the use of diagnostic
tests could reduce antibiotic prescription rates. The objective of this study was
to evaluate whether access to a multiplex polymerase chain reaction (PCR) assay panel
for etiologic diagnosis of acute respiratory tract infections (ARTIs) would have an
impact on antibiotic prescription rate in primary care clinical settings.

Methods

Adult patients with symptoms of ARTI were prospectively included. Nasopharyngeal and
throat swabs were analysed by using a multiplex real-time PCR method targeting thirteen
viruses and two bacteria. Patients were recruited at 12 outpatient units from October
2006 through April 2009, and samples were collected on the day of inclusion (initial
visit) and after 10 days (follow-up visit). Patients were randomised in an open-label
treatment protocol to receive a rapid or delayed result (on the following day or after
eight to twelve days). The primary outcome measure was the antibiotic prescription
rate at the initial visit, and the secondary outcome was the total antibiotic prescription
rate during the study period.

Results

A total sample of 447 patients was randomised. Forty-one were excluded, leaving 406
patients for analysis. In the group of patients randomised for a rapid result, 4.5%
(9 of 202) of patients received antibiotics at the initial visit, compared to 12.3%
(25 of 204) (P = 0.005) of patients in the delayed result group. At follow-up, there was no significant
difference between the groups: 13.9% (28 of 202) in the rapid result group and 17.2%
(35 of 204) in the delayed result group (P = 0.359), respectively.

Conclusions

Access to a rapid method for etiologic diagnosis of ARTIs may reduce antibiotic prescription
rates at the initial visit in an outpatient setting. To sustain this effect, however,
it seems necessary to better define how to follow and manage the patient according
to the result of the test, which warrants further investigation.

Trial registration

Background

Acute respiratory tract infections (ARTIs) represent a major global health burden
[1], and viruses cause a large proportion of ARTIs. Distinguishing bacterial ARTIs that
require antibiotic treatment from viral ARTIs not needing an antibiotic prescription
can be difficult on clinical grounds alone and causes unnecessary use of antibiotics,
with the highest rates occurring in the primary care setting [2,3]. Excess use of antibiotics has major implications for health economics and, more
importantly, for the development of bacterial resistance [3,4], as well as for the individual patient in terms of adverse events such as allergic
reactions and antibiotic-associated diarrhoea [5,6]. The predictive value of vital signs, C-reactive protein (CRP) and X-ray findings
for diagnosing pneumonia requiring antibiotics is low [7,8]. The treatment of acute bronchitis serves as an illustrative example of the unnecessary
use of antibiotics, where the recommended therapy for immune competent adults does
not include antibiotic treatment [9,10], yet antibiotics were found to have been prescribed for this condition at high rates
in studies conducted in the United Kingdom (64%) [11], Sweden (50% to 60%) [12,13] and the United States (59%) [14].

The use of improved diagnostic methods such as nucleic acid amplification tests (NAATs),
including multiplex real-time polymerase chain reaction (RT-PCR) assays, has increased
in recent years. These methods have proven to be equivalent or superior to conventional
methods [15-19] and to have a short turnaround time at the laboratory, as well as affording clinicians
the ability to analyse several respiratory agents within the same patient sample.

It has been suggested that the use of NAATs, including multiplex PCR methods, for
the detection of respiratory pathogens could reduce antibiotic prescription rates
[14,18]. The present study was designed to evaluate whether access to a multiplex RT-PCR
method targeting thirteen viruses would have an impact on antibiotic prescription
rates for ARTI in a primary care setting.

Methods

Study design

We conducted an investigator-initiated, multicentre, prospective, randomised, controlled
trial with adult patients in a primary care setting. Eligibility criteria for participants
were age ≥ 18 years and a diagnosis of community-acquired ARTI, defined as having
a history of at least two of the following symptoms: coryza/nasal congestion/sneezing,
sore throat/odynophagia, cough, pleuritic chest pain, shortness of breath or fever
for which the physician found no other explanation, with a duration of less than 14
days. Exclusion criteria included confirmed bacterial infection (defined as a positive
Streptococcus group A quick test and clinical findings corresponding to bacterial tonsillitis, perforated
acute otitis media, high suspicion of lobar pneumococcal pneumonia or severe septicaemia,
a positive blood culture for a clinically significant bacterial pathogen and clinical
findings corresponding to septicaemia) and ongoing antibiotic treatment. Patients
were recruited at 12 outpatient units (eight primary healthcare centres and four departments
of infectious diseases), and samples were collected from October 2006 through April
2009. Signs and symptoms were recorded in a web-based case report form.

Randomisation and masking

Patients were enrolled by the treating physician on the day of inclusion and stratified
according to duration of symptoms of either ≤5 days or > 5 days. Open-label (nonblinded)
randomisation (ratio 1:1) was performed by means of a predefined list and using a
concealed, central, web-based procedure on the day of inclusion for the treating physician
to receive the results from the multiplex PCR analysis either on the day following
inclusion (the rapid result cohort) or eight to twelve days later (the delayed result
cohort).

Recruitment was performed from Sunday through Thursday from 8 AM until 5 PM, allowing
for the laboratory to report results the following day. Nasopharyngeal (flocked nylon
swabs; Microrheologics, Brescia, Italy) and throat swab specimens were collected from
each patient. The swabs were jointly placed in a sterile container with 1 mL of sodium
chloride solution and sent to the laboratory the same day. Specimens were either analyzed
directly or frozen at -70°C for delayed analysis (see randomisation and masking above).
The results were communicated through the web-based case report form to a study nurse
at each site. Additional diagnostic testing, including throat and sputum cultures,
CRP and X-ray investigations, were left to the discretion of the treating physician
and recorded in the case report form.

Outcome measures

The objective of the study was to evaluate whether access to a rapid etiologic diagnostic
method would have an impact on antibiotic prescription. The primary outcome measure
was the antibiotic prescription rate at the initial visit (or within 48 hours thereafter),
and the secondary outcome measure was the total antibiotic prescription rate during
the study period. Antibiotic prescription at the initial visit (or within 48 hours
thereafter) was recorded and analysed in relation to access to a rapid vs. a delayed
result. Results in the rapid result group were provided to the treating physician
within 24 hours for the majority of patients and within 48 hours for all patients.
The final management of all patients and how to act upon the given result of the PCR
assay were left to the discretion of the treating physician. All patients were asked
to return for a follow-up visit eight to twelve days after the initial visit, and
this time period represents the duration of follow-up in the study. The total antibiotic
prescription rate (prescriptions at initial visit, at follow-up visit or between those
visits) was recorded and constituted the secondary outcome measure in the study. The
prescription of antiviral medications was not recorded. Serious adverse events (SAEs)
were defined as death, life-threatening events, hospitalisation or events resulting
or threatening to result in persistent or significant disability. The Regional Ethical
Review board approved the study, and all patients provided written informed consent
to participate in the study.

Nucleic acid extraction and RT-PCR

We utilized a RT-PCR procedure based on automated specimen extraction and multiplex
amplification adapted for respiratory specimens as previously described [20]. Briefly, nucleic acid from 100 μL of the respiratory specimen was extracted into
an elution volume of 100 μL by using a Magnapure LC robot (Roche Molecular Systems,
Mannheim, Germany) according to the total nucleic acid protocol and was amplified
in an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) in
50-μL reaction volumes. After a reverse transcription step, 45 cycles of two-step
PCR were performed. Each sample was amplified in six parallel reactions containing
primers and probes as previously reported [20], with the modification of adenovirus being analysed in a separate reaction. Included
in the panel were parainfluenzavirus (PIV) types 1 through 3, influenza virus A (IfA)
and influenza virus B (IfB), human metapneumovirus, respiratory syncytial virus (RSV),
human rhinovirus (hRV), enterovirus (EV), adenovirus (AdV) and human coronavirus (hCoV)
types 229E, OC43 and NL63, along with the bacteria Mycoplasma pneumoniae and Chlamydophila pneumoniae.

The primers and probes of all RT-PCR assays were designed to bind conserved segments
of the targeted agents. This is particularly important for AdV, hRV and EV, which
are characterised by a large number of subtypes. The accuracy of the AdV PCR assay
has been documented by Heim et al. [21]. The target region for the hRV and EV assays was the conserved segment of the 5'
untranslated region that allows amplification of all subtypes and which has been used
previously by others [22-24]. The primers and probes used for IfB and PIV type 3 were developed by Dr Lars Nielsen,
Copenhagen, Denmark; those for PIV types 1 and 2 were previously described by Watzinger
et al. [22]; those for hCoV (types NL63, 229E and OC43) were described previously by Gunson et al. [24]; and those for IfA virus were a modification of a system published by Ward et al. [25].

Statistical analysis

On the basis of the nature of the primary outcome measure, patients with protocol
violations and/or missing data were excluded from the primary analysis. This was predefined
in the analysis plan. The χ2 test was used to compare proportions. P < 0.05 was considered statistically significant. The study was scheduled to include
at least 200 patients in each group, allowing for a statistical power of 80% to demonstrate
an estimated reduction in antibiotic prescription rate from 20% to 10% in the rapid
result group. Multivariate analysis using backward stepwise (Wald test) logistic regression
was carried out to analyse factors separately and independently to predict a positive
PCR result as well as the prescription of antibiotics. SPSS version 17.0 for Macintosh
software (SPSS Inc, Chicago, IL, USA) was used for all statistical analyses.

Results

Study design and baseline characteristics

The patient flow according to the study design is shown in Figure 1. The baseline characteristics of the two groups of patients randomised for rapid
vs. delayed results are shown in Table 1.

Primary outcome measure

In the entire study population, antibiotics were prescribed for 8.4% (34 of 406) of
patients at the initial visit (or within 48 hours thereof). In the group of patients
randomised for a rapid result, 4.5% (9 of 202) of patients received an antibiotic,
compared to 12.3% (25 of 204) of patients in the delayed PCR-based result group (P = 0.005) (see Table 2). Patients with symptom duration ≤ 5 days in the rapid result group received significantly
fewer antibiotic prescriptions than patients in the delayed result group.

Of the 34 patients who received initial antibiotic treatment, 14 (41%) tested positive
for a respiratory virus, comprising three in the rapid result group and eleven in
the delayed result group (see Table 2).

Secondary outcome measure

A total of 335 (83%) of 406 patients returned for the optional follow-up visit or
were available for a telephone appointment (visit, n = 243; telephone appointment, n = 92), comprising 166 (82%) of 202 patients in the rapid result group and 169 (83%)
of 204 patients in the delayed result group. In total, 28 patients (13.9%) in the
rapid result group and 35 patients (17.2%) in the delayed result group received antibiotic
treatment at either the initial or follow-up visit. This difference was not statistically
significant (P = 0.359). Antibiotic prescriptions outside the study (that is, by other than the study
physician) were allowed. At the follow-up visit, two (11%) of nineteen patients in
the rapid result group and one (11%) of nine patients in the delayed result group
reported ongoing antibiotic treatment prescribed outside the study. The investigators
reported no SAEs.

Aetiology

As shown in Table 3, 191 patients (47%) tested positive for one agent on the basis of a multiplex PCR
assay performed at the initial visit. In addition, 12 patients (5.9%) tested positive
for two agents in the same sample. In three of these patients, one virus and one bacterium
were detected, and in nine patients, two viruses were detected (see Table 4).

Table 3. Results (multiple detections not included) of multiplex real-time polymerase chain
reaction assays of all included patients in order of frequency and by randomisation
group (rapid vs. delayed result group)

Table 4. Codetection of agents in multiplex real-time polymerase chain reaction assays from
the same nasopharyngeal/oropharyngeal sample at initial visit of adults with ARTIa by randomisation group (rapid result vs. delayed result)

CRP levels

CRP levels were recorded in 301 (74%) of 406 patients, and a predefined subgroup analysis
revealed that 39 patients (13%) had a CRP level ≥50 mg/L and that 15 patients (5%)
had a CRP level > 100 mg/L. Of the 39 patients with a CRP level ≥50 mg/L, 12 patients
(31%) received antibiotic treatment at the initial visit, compared to 17 (7%) of 262
patients in the group with a CRP level < 50 mg/L (P < 0.0001). In the rapid result group, two (8%) of twenty-four patients with a CRP
level ≥50 mg/L received antibiotics, compared to 10 (67%) of 15 patients in the delayed
result group (P = 0.0001). Patients who tested positive for Mycoplasma pneumoniae or Chlamydophila pneumoniae were included in the subgroup analysis of CRP levels (values for M. pneumoniae, available for five of seven patients, ranged from 58 mg/L to 210 mg/L, and the CRP
level was 35 mg/L for the only patient who tested positive for C. pneumoniae). A virus (M. pneumoniae and C. pneumoniae excluded) was found in 49% of patients (19 of 39) with a CRP level ≥50 mg/L and in
27% of patients (4 of 15) with a CRP level ≥100 mg/L.

Vomiting remained the only symptom associated with the prescription of antibiotics
at the initial visit (OR 5.91, 95% CI 2.20 to 15.85, P = 0.0004) in multivariate analysis. High fever (≥38.5°C) was more common in the group
of patients randomised for a delayed result (12 of 201 patients; 6.0%) than in the
group randomised for a rapid result (6 of 198; 3.0%) (see Table 1), but no significant difference in the number of antibiotic prescriptions at the
initial visit for these groups was noted (see Table 2).

Discussion

We have shown that access to a rapid result using a method for aetiologic diagnosis
of ARTIs in an outpatient setting significantly reduced antibiotic prescriptions at
the initial visit. However, this effect was no longer significant at the time of follow-up.
In our study, we evaluated the impact of access to a rapid diagnostic tool rather
than the impact of the actual test result, which implies that the mere prospect of
a rapid aetiologic diagnosis can influence therapeutic decisions made for patients
with an indistinct clinical presentation.

In the subgroup of patients with a CRP level ≥50 mg/L, this effect was even more pronounced.
Among patients with positive PCR results for viruses, significantly fewer patients
in the rapid result group received antibiotics than in the delayed result group. Our
results are in line with the reduction of antibiotic prescriptions when rapid diagnostic
tests for IfA virus were used systematically for hospitalised adult patients [26] and children [27], although these studies evaluated the impact of the result of the test and thus are
not fully comparable.

The limitations of this study include the choice of antibiotic prescription as the
primary outcome and its open-label design, which might have led to performance bias;
that is, the physicians might have been influenced by the randomisation in making
the decision whether to prescribe antibiotics. Also, on the basis of the study design
with an optional follow-up visit, a large number of patients were lost to follow-up,
and because of the relatively short follow-up period, the effect and duration of antibiotic
treatment in relation to the diagnosis could not be properly evaluated. To influence
antibiotic resistance and adverse events following antibiotic therapy, it is necessary
to reduce the total rate of antibiotic prescriptions, which was not achieved in our
study. However, at the time of planning for the study, limited prospective data were
available on the performance of a multiplex PCR panel for the diagnosis of viral and
bacterial ARTIs in clinical practise. It was not possible to define whether patients
should be prescribed an antibiotic depending on the test result. We therefore chose
antibiotic prescriptions at the initial visit as a straightforward primary outcome
measure to evaluate whether access to the test would have any effect on antibiotic
prescription rates, and this should be included in future studies of algorithms for
the management of patients with ARTIs.

Systematic testing for bacterial pathogens such as Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis was not conducted in this study. These bacteria may act as potential respiratory pathogens
(PRPs) harbouring in the upper respiratory tract, and an extension of the panel to
include these bacterial agents might improve the clinical utility of the test. However,
the interpretation of the detection of bacterial pathogens in nasopharyngeal samples
in heterogeneous syndromes such as ARTIs is unclear. Cultured S. pneumoniae from the nasopharynx of adults has been shown to have high specificity but low sensitivity
for the diagnosis of pneumococcal pneumonia [28]. PCR-based detection of S. pneumoniae from the oropharynx and nasopharynx without codetection of other commensal Streptococcus spp. poses technical difficulties.

Lieberman et al. [29] reported higher sensitivity in using nasopharyngeal washing compared to nasopharyngeal
swabs in the detection of PRPs. To keep sampling simple, nasopharyngeal washing was
not used in this study, which may constitute a study limitation. However, we used
flocked nasopharyngeal swabs, which probably yield higher detection rates than previously
reported in studies in which cotton-tipped nasopharyngeal swabs were used [30]. The study population consisted of patients with a broad spectrum of clinical manifestations,
many of whom had mild symptoms. However, since the prescription of antibiotics for
these patients is not uncommon, this was a deliberate study design.

Previously, Oosterheert et al. [18] investigated the impact of a PCR method for aetiologic diagnosis of lower RTIs in
adults but failed to show any reduction in the use of antibiotics. However, their
study included only hospitalised patients, and the end point was to register a change
of ongoing treatment regimen rather than whether to make the initial decision to prescribe
antibiotic treatment.

At follow-up, our study no longer showed any significant difference in antibiotic
use between the two study groups. However, since a large number of patients were lost
to follow-up, the selection of patients may have been biased. Moreover, physicians
outside the study prescribed antibiotics for some patients between the initial visit
and the follow-up visit. Because of the study design, the accuracy of prescriptions
during follow-up could not be properly evaluated. Thus, our results must be interpreted
with caution. In the absence of an algorithm to determine how to follow the patients
and act upon the results of PCR testing, including a predefined antibiotic management
plan, the observed reduction of antibiotic use at the initial visit may be lost by
the time of follow-up. However, our study represents a proof of concept that access
to a rapid etiologic diagnostic tool may affect therapeutic decision in this setting.

The antibiotic prescription rate in our study was low. A recent retrospective study
of primary care patients in a comparable Swedish region recorded an antibiotic prescription
rate of 45% for all patients with ARTIs, 60% for patients with acute bronchitis and
16% for patients with the 'common cold' [13]. The low prescription rate in our series could be due partly to a tendency to adhere
more strictly than usual to current guidelines for the restrictive use of antibiotics.

We detected an infectious agent in 47% of patient samples taken at the initial visit,
which is in line with previous studies in which detection rates between 43% and 63%
in adults have been described [11,31]. Detection of a virus does not exclude concomitant bacterial infections or other
noninfectious causes. This safety issue was discussed at the beginning of our study,
and physicians were encouraged to treat bacterial complications at their own discretion.
No SAEs were reported, but because of the relatively large number of patients lost
to follow-up and the short duration of the study, we cannot exclude the possibility
that some patients experienced late SAEs. Patient-relevant outcomes, such as the severity
of symptoms over time, were not recorded, which is another limitation of this study.
We deliberately chose not to include such outcomes, as the primary objective of the
study was to evaluate the impact on antibiotic prescription rates at the initial visit.
This study design also reduced the resources needed to conduct the study.

Analysis of CRP is frequently used to discriminate viral from bacterial infections
[32]. Prescription rates increase with rising CRP levels [32,13]. In the group of patients with CRP levels > 100 mg/L, approximately one-third carried
a respiratory virus, supporting the need for reliable tools for the aetiologic diagnosis
of ARTIs. Distinguishing a viral from a bacterial aetiology in ARTIs is difficult
on clinical grounds alone, and the predictive value of vital signs, CRP and X-ray
findings is low [7,8]. Procalcitonin (PCT) has been proven to be useful in reducing antibiotic prescriptions
in hospitalised patients with lower RTIs [33,34], whereas in primary care, the evidence of PCT for this purpose is more ambiguous.
One study of adults with ARTI (one to twenty-eight days' illness duration) judged
to be in need of antibiotics and treated as outpatients reported a positive effect
of antibiotic use [35], but another study of children with community-acquired pneumonia, a significant proportion
of whom were treated in the hospital, showed a negative effect [36].

No distinct pattern of symptoms that could guide the clinician towards a correct aetiologic
diagnosis was identified in our study. Vomiting, which was the only independent variable
predicting antibiotic use, may be interpreted as a sign of serious illness justifying
antibiotic treatment.

Reducing the unnecessary use of antibiotics in the treatment of patients with ARTIs
is of utmost importance and cannot be accomplished by using a single strategy. Patient-
and physician-oriented educational programmes could play an important role as previously
suggested [37]. However, these programmes do not solve the issue of the lack of an aetiologic diagnosis,
which is important not only for adequate use of antibiotics but also for addressing
issues such as possible complications, prognosis, antiviral treatment options, surveillance
and infection control.

Conclusions

In conclusion, we have shown that access to a multiplex RT-PCR assay for the aetiologic
diagnosis of ARTIs may reduce the prescription rate of antibiotics at the initial
visit in an outpatient setting. To sustain the effect, it seems necessary to define
how to follow and manage the patient according to the result of the test, which warrants
further investigation. We believe that the implementation of similar methods in routine
clinical care may be a useful tool to reduce the overprescription of antibiotics in
patients with ARTIs.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

RBL contributed to the study design, data collection, data analysis and the writing
of the manuscript and was the main person responsible for database management. JW
and LMA contributed to the study design, data collection, data analysis and the writing
of the manuscript. SO and ML contributed to the study design, technical issues and
the writing of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors acknowledge the staff at the Clinical Virology Laboratory, Department
of Virus Detection, at Sahlgrenska University Hospital for their technical expertise,
as well as the patients and staff at the following centres in the Region Västra Götaland:
the Departments of Infectious Diseases in Uddevalla, Skövde, Borås and Göteborg, as
well as staff at primary health care centres in Stenungsund, Sollebrunn, Floda, Kungshöjd,
Kungsten, Olskroken and Carlanderska, as well as Capio Axess Akuten. This study was
funded by the Swedish Strategic Programme against Antibiotic Resistance (Strama),
Capio Research Foundation, grant 2006-1166, and Region Västra Götaland grant VGFOUREG-8402.